DOCSLIB.ORG

Stress and Interferon Signalling-Mediated Apoptosis Contributes to Pleiotropic Anticancer Responses Induced by Targeting NGLY1

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Stress and Interferon Signalling-Mediated Apoptosis Contributes to Pleiotropic Anticancer Responses Induced by Targeting NGLY1 www.nature.com/bjc ARTICLE Cellular and Molecular Biology Stress and interferon signalling-mediated apoptosis contributes to pleiotropic anticancer responses induced by targeting NGLY1 Ashwini Zolekar1, Victor. J. T. Lin1, Nigam M. Mishra1, Yin Ying Ho2, Hamed S. Hayatshahi1, Abhishek Parab3, Rohit Sampat1, Xiaoyan Liao4,6, Peter Hoffmann2,5, Jin Liu1, Kyle A. Emmitte1 and Yu-Chieh Wang1 BACKGROUND: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. METHODS: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. RESULTS: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. CONCLUSION: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy. British Journal of Cancer (2018) 119:1538–1551; https://doi.org/10.1038/s41416-018-0265-9 BACKGROUND congenital deglycosylation disorder, were recently identified.2,5,8,9 As a pivotal glycosidase known for catalysing the removal of Many of these mutations cause premature termination of glycans from N-glycosylated asparagine residues, NGLY1 (a.k.a. translation, leading to complete loss of NGLY1 in the patients. PNGase) enables the deglycosylation of denatured glycoproteins Until this discovery, the disease implications of NGLY1 had not and allows proteasome-mediated protein degradation to effi- been definitive. ciently occur.1–5 A TGase-superfamily (PNGase-core) domain exists NGLY1 deficiency causes a broad spectrum of disease in NGLY1 proteins ranging from yeast to human,4 suggesting the phenotypes with incomplete penetrance in patients.2,5,8,9 Many evolutionarily conserved significance of NGLY1 enzymatic activity NGLY1 deficiency-associated phenotypes are closely related to in cells. It is known that loss of NGLY1 function in cells can cause developmental delay and congenital abnormalities, suggesting the accumulation of aberrant proteins in the cytosol and the the significant role and intricate regulation of this glycosidase in interruption of endoplasmic reticulum-associated protein degra- the normal development of human organs. Despite the recently dation (ERAD).1,4,5 Therefore, NGLY1 defects are likely to affect the gained knowledge about NGLY1 deficiency, there is limited quality control and homeostasis of many cellular proteins, information regarding the responses of human cancer cells and subsequently perturbing signalling pathways, cell physiology, terminally differentiated somatic cells to NGLY1 suppression. and organ development. The studies of an NGLY1 ortholog gene, NGLY1 is commonly expressed in many types of normal and PNGase-like (Pngl), in fruit fly and fungus also indicate that NGLY1 cancer cells (www.proteinatlas.org),10 suggesting that NGLY1 could be involved in the regulation of cell normality through an could be essential for a variety of human cells regardless of their enzymatic activity-independent mechanism.6,7 Interestingly, pathophysiological conditions. Notably, NGLY1 appears to be human NGLY1 gene mutations that result in NGLY1 deficiency, a highly expressed in certain human cancer cells (e.g., melanoma 1Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX, USA; 2Adelaide Proteomics Centre, The University of Adelaide, Adelaide, Australia; 3Department of Mathematics, Purdue University, West Lafayette, Indiana, USA; 4Department of Pathology, University of California, San Diego, San Diego, CA, USA and 5Future Industries Institute, University of South Australia, Adelaide, Australia Correspondence: Y-C. Wang ([email protected]) 6Present address: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA These authors contributed equally: Ashwini Zolekar, Victor J.T. Lin. Received: 26 April 2018 Revised: 11 August 2018 Accepted: 31 August 2018 Published online: 2 November 2018 © The Authors 2018 Published by Springer Nature on behalf of Cancer Research UK Stress and interferon signalling-mediated apoptosis contributes to. A Zolekar et al. 1539 and ovarian cancer), while low-to-undetectable in their normal Knockdown of NGLY1 and GADD153 counterpart tissues (e.g., skin and ovary) (www.proteinatlas.org).10 The knockdown of NGLY1 expression in melanoma cells was These observations raise an intriguing possibility that NGLY1 may achieved by the transduction of pZIP-TRE3GS lentiviral expression be crucial for cancer development and progression. Moreover, vectors that carry two independent shRNA sequences (Supple- cancer cells, at least in certain cancer types, may be particularly mentary Materials and Methods; TransOMIC Technologies, Hunts- vulnerable to loss of NGLY1 compared with normal cells. ville, AL). A pZIP-TRE3GS vector that carries a NT-shRNA sequence In this study, we systematically and mechanistically examined was used as the control. The expression of the shRNA sequences the significance of NGLY1 in melanoma cells and how it may be and an open reading frame of the ZsGreen reporter is driven exploited as a novel anticancer target. In addition, we developed by the TRE3GS doxycycline-inducible promoter. The transduced and tested novel covalent inhibitors that suppress NGLY1 activity cells were selected using puromycin for a prolonged period in human cells. Our results strongly support that (~4 weeks) to obtain the stable clones of cancer cells that carry NGLY1 suppression in melanoma cells elicits multifaceted inducible NT-shRNA, NGLY1-shRNA645 and NGLY1-shRNA647 cancer-elimination responses, and that targeting NGLY1 and sequences. protein deglycosylation may represent a novel anticancer strategy The knockdown of GADD153 expression in melanoma cells with the opportunity for a broad therapeutic window. was achieved by the transduction of pZIP-hEF1a-RFP lentiviral expression vectors that carry three independent shRNA sequences (Supplementary Materials and Methods; TransOMIC MATERIALS AND METHODS Technologies, Huntsville, AL). A pZIP-hEF1a-RFP lentiviral Cell Culture expression vector carries a NT-shRNA sequence was used as Human dermal fibroblasts were cultured in DMEM (Thermo Fisher the control. The expression of the shRNA sequences and an Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS; open reading frame of the RFP reporter is driven by the human Thermo Fisher Scientific, Carlsbad, CA) at 37°C. HEMl and HEMd EF1α gene promoter. (ScienCell Research Laboratories, Carlsbad, CA) cells were cultured in melanocyte medium (MelM; ScienCell Research Laboratories, Overexpression of human NGLY1 Carlsbad, CA). Human melanoma cells were cultured using RPMI- A pLenti expression vector that carries a Myc-DDK-tagged-human 1640 medium (Thermo Fisher Scientific, Carlsbad, CA) or DMEM/ NGLY1 open reading frame driven by a CMV promoter (OriGene F12 medium (Thermo Fisher Scientific, Carlsbad, CA) containing Technologies, Rockville, MD) was transduced into cells for the 1234567890();,: 10% FBS. WA09 human embryonic stem cells (hESCs) were overexpression of NGLY1. A pLenti-C-Myc-DDK empty vector was obtained from the WiCell Stem Cell Bank (WiCell Research used as the transduction control. Institute, Madison, WI). HMi-50611 and NGLY1Pt1i-509 hiPSCs were established using CytoTune Sendai Reprogramming Kit Immunohistochemistry (IHC) and Fluorescence Staining (Thermo Fisher Scientific, Carlsbad, CA). We followed the The general procedure for antibody-mediated fluorescence previously described method12 for culturing undifferentiated staining was previously described12 and provided as part human pluripotent stem cells (hPSCs) in a feeder cell-free of Supplementary Materials and Methods. The detailed informa- condition, except the use of TeSR-E8 medium (Stemcell Technol- tion of primary antibodies was summarised in Supplementary ogies, Vancouver, Canada) and L7 hPSC passaging solution (Lonza, Table S2. Walkersville, MD) in this study. The detailed information of cells used in this study was summarised in Supplementary Table S1. Immunoblotting The experiments using hPSCs were performed in compliance with The general procedure for immunoblotting was described in a the guidelines and approval of the institutional biosafety previously published report,13 except that cell lysates were committee at UNTHSC. All cells were periodically tested using prepared using M-PER mammalian protein extraction
Load more

Recommended publications
  • 2020 Program Book
    PROGRAM BOOK Note that TAGC was cancelled and held online with a different schedule and program. This document serves as a record of the original program designed for the in-person meeting. April 22–26, 2020 Gaylord National Resort & Convention Center Metro Washington, DC TABLE OF CONTENTS About the GSA ........................................................................................................................................................ 3 Conference Organizers ...........................................................................................................................................4 General Information ...............................................................................................................................................7 Mobile App ....................................................................................................................................................7 Registration, Badges, and Pre-ordered T-shirts .............................................................................................7 Oral Presenters: Speaker Ready Room - Camellia 4.......................................................................................7 Poster Sessions and Exhibits - Prince George’s Exhibition Hall ......................................................................7 GSA Central - Booth 520 ................................................................................................................................8 Internet Access ..............................................................................................................................................8
    [Show full text]
  • 2021 Western Medical Research Conference
    Abstracts J Investig Med: first published as 10.1136/jim-2021-WRMC on 21 December 2020. Downloaded from Genetics I Purpose of Study Genomic sequencing has identified a growing number of genes associated with developmental brain disorders Concurrent session and revealed the overlapping genetic architecture of autism spectrum disorder (ASD) and intellectual disability (ID). Chil- 8:10 AM dren with ASD are often identified first by psychologists or neurologists and the extent of genetic testing or genetics refer- Friday, January 29, 2021 ral is variable. Applying clinical whole genome sequencing (cWGS) early in the diagnostic process has the potential for timely molecular diagnosis and to circumvent the diagnostic 1 PROSPECTIVE STUDY OF EPILEPSY IN NGLY1 odyssey. Here we report a pilot study of cWGS in a clinical DEFICIENCY cohort of young children with ASD. RJ Levy*, CH Frater, WB Galentine, MR Ruzhnikov. Stanford University School of Medicine, Methods Used Children with ASD and cognitive delays/ID Stanford, CA were referred by neurologists or psychologists at a regional healthcare organization. Medical records were used to classify 10.1136/jim-2021-WRMC.1 probands as 1) ASD/ID or 2) complex ASD (defined as 1 or more major malformations, abnormal head circumference, or Purpose of Study To refine the electroclinical phenotype of dysmorphic features). cWGS was performed using either epilepsy in NGLY1 deficiency via prospective clinical and elec- parent-child trio (n=16) or parent-child-affected sibling (multi- troencephalogram (EEG) findings in an international cohort. plex families; n=3). Variants were classified according to Methods Used We performed prospective phenotyping of 28 ACMG guidelines.
    [Show full text]
  • A Drosophila Natural Variation Screen Identifies NKCC1 As a Substrate of NGLY1 Deglycosylation and a Modifier of NGLY1 Deficienc
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.13.039651; this version posted April 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Title 2 A Drosophila natural variation screen identifies NKCC1 as a substrate of NGLY1 deglycosylation 3 and a modifier of NGLY1 deficiency 4 Authors 5 Dana M. Talsness1, Katie G. Owings1, Emily Coelho1, Gaelle Mercenne2, John M. Pleinis2, Aamir 6 R. Zuberi3, Raghavendran Partha4, Nathan L. Clark1, Cathleen M. Lutz3, Aylin R. Rodan2,5, 7 Clement Y. Chow1* 8 The first two authors contributed equally to this study. 1Department of Human Genetics, University 9 of Utah School of Medicine, Salt Lake City, UT; 2Department of Internal Medicine, Division of 10 Nephrology and Hypertension, and Molecular Medicine Program, University of Utah, Salt Lake 11 City, UT; 3Genetic Resource Science, The Jackson Laboratory, Bar Harbor, ME.; 4Department of 12 Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA; 5Medical Service, 13 Veterans Affairs Salt Lake City Health Care System, Salt Lake City, UT; *For correspondence: 14 [email protected] 15 Abstract 16 N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in 17 the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, 18 seizures, and a lack of sweat and tears. To model the phenotypic variability observed among 19 patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse 20 strains.
    [Show full text]
  • Mouse Ngly1 Conditional Knockout Project (CRISPR/Cas9)
    https://www.alphaknockout.com Mouse Ngly1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ngly1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ngly1 gene (NCBI Reference Sequence: NM_021504 ; Ensembl: ENSMUSG00000021785 ) is located on Mouse chromosome 14. 12 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000022310). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ngly1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-304N3 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit dysregulation of the endoplasmic reticulum (ER)-associated degradation (ERAD) process. Exon 2 starts from about 6.76% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 5144 bp, and the size of intron 2 for 3'-loxP site insertion: 5807 bp. The size of effective cKO region: ~615 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ngly1 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats.
    [Show full text]
  • Regulation of BMP4/Dpp Retrotranslocation and Signaling By
    RESEARCH ADVANCE Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation Antonio Galeone1,2, Joshua M Adams3,4, Shinya Matsuda5, Maximiliano F Presa6, Ashutosh Pandey1, Seung Yeop Han1, Yuriko Tachida7,8, Hiroto Hirayama7,8, Thomas Vaccari2, Tadashi Suzuki7,8, Cathleen M Lutz6, Markus Affolter5, Aamir Zuberi6, Hamed Jafar-Nejad1,3* 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States; 2Department of Biosciences, University of Milan, Milan, Italy; 3Program in Developmental Biology, Baylor College of Medicine, Houston, United States; 4Medical Scientist Training Program, Baylor College of Medicine, Houston, United States; 5Biozentrum, University of Basel, Basel, Switzerland; 6The Jackson Laboratory, Bar Harbor, United States; 7Glycometabolome Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Saitama, Japan; 8T-CiRA joint program, Kanagawa, Japan Abstract During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its *For correspondence: retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the [email protected] ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing Competing interests: The properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling authors declare that no competing interests exist.
    [Show full text]
  • ER-Resident Transcription Factor Nrf1 Regulates Proteasome Expression and Beyond
    International Journal of Molecular Sciences Review ER-Resident Transcription Factor Nrf1 Regulates Proteasome Expression and Beyond Jun Hamazaki * and Shigeo Murata Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo 1130033, Japan; [email protected] * Correspondence: [email protected] Received: 4 May 2020; Accepted: 20 May 2020; Published: 23 May 2020 Abstract: Protein folding is a substantively error prone process, especially when it occurs in the endoplasmic reticulum (ER). The highly exquisite machinery in the ER controls secretory protein folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol; these misfolded proteins are then degraded by the ubiquitin–proteasome system termed as the ER-associated degradation (ERAD). The 26S proteasome is a multisubunit protease complex that recognizes and degrades ubiquitinated proteins in an ATP-dependent manner. The complex structure of the 26S proteasome requires exquisite regulation at the transcription, translation, and molecular assembly levels. Nuclear factor erythroid-derived 2-related factor 1 (Nrf1; NFE2L1), an ER-resident transcription factor, has recently been shown to be responsible for the coordinated expression of all the proteasome subunit genes upon proteasome impairment in mammalian cells. In this review, we summarize the current knowledge regarding the transcriptional regulation of the proteasome, as well as recent findings concerning the regulation of Nrf1 transcription activity in ER homeostasis and metabolic processes. Keywords: proteasome; Nrf1; DDI2; bounce back; proteostasis 1. Introduction Proteins are essential to the vast majority of cellular and organismal functions. Cellular protein levels are defined by the balance among protein synthesis, folding, and degradation, and the failure to accurately coordinate these processes leads to disease [1].
    [Show full text]
  • A SARS-Cov-2 Protein Interaction Map Reveals Targets for Drug Repurposing
    Article A SARS-CoV-2 protein interaction map reveals targets for drug repurposing https://doi.org/10.1038/s41586-020-2286-9 A list of authors and affiliations appears at the end of the paper Received: 23 March 2020 Accepted: 22 April 2020 A newly described coronavirus named severe acute respiratory syndrome Published online: 30 April 2020 coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than Check for updates 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efcacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and eforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identifed the human proteins that physically associated with each of the SARS-CoV-2 proteins using afnity-purifcation mass spectrometry, identifying 332 high-confdence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors.
    [Show full text]
  • Human Tagged ORF Clone Lentiviral Particle – RC227451L3V
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC227451L3V NGLY1 (NM_001145295) Human Tagged ORF Clone Lentiviral Particle Product data: Product Type: Lentiviral Particles Product Name: NGLY1 (NM_001145295) Human Tagged ORF Clone Lentiviral Particle Symbol: NGLY1 Synonyms: CDDG; CDG1V; PNG-1; PNG1; PNGase Vector: pLenti-C-Myc-DDK-P2A-Puro (PS100092) ACCN: NM_001145295 ORF Size: 1674 bp ORF Nucleotide The ORF insert of this clone is exactly the same as(RC227451). Sequence: OTI Disclaimer: The molecular sequence of this clone aligns with the gene accession number as a point of reference only. However, individual transcript sequences of the same gene can differ through naturally occurring variations (e.g. polymorphisms), each with its own valid existence. This clone is substantially in agreement with the reference, but a complete review of all prevailing variants is recommended prior to use. More info OTI Annotation: This clone was engineered to express the complete ORF with an expression tag. Expression varies depending on the nature of the gene. RefSeq: NM_001145295.1, NP_001138767.1 RefSeq Size: 2421 bp RefSeq ORF: 1677 bp Locus ID: 55768 UniProt ID: Q96IV0 MW: 63.8 kDa Gene Summary: This gene encodes an enzyme that catalyzes hydrolysis of an N(4)-(acetyl-beta-D- glucosaminyl) asparagine residue to N-acetyl-beta-D-glucosaminylamine and a peptide containing an aspartate residue. The encoded enzyme may play a role in the proteasome- mediated degradation of misfolded glycoproteins. Multiple transcript variants encoding different isoforms have been found for this gene.[provided by RefSeq, Feb 2009] This product is to be used for laboratory only.
    [Show full text]
  • NGLY1 (NM 001145295) Human Tagged ORF Clone Product Data
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC227451L4 NGLY1 (NM_001145295) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: NGLY1 (NM_001145295) Human Tagged ORF Clone Tag: mGFP Symbol: NGLY1 Synonyms: CDDG; CDG1V; PNG-1; PNG1; PNGase Vector: pLenti-C-mGFP-P2A-Puro (PS100093) E. coli Selection: Chloramphenicol (34 ug/mL) Cell Selection: Puromycin ORF Nucleotide The ORF insert of this clone is exactly the same as(RC227451). Sequence: Restriction Sites: SgfI-MluI Cloning Scheme: ACCN: NM_001145295 ORF Size: 1674 bp This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 2 NGLY1 (NM_001145295) Human Tagged ORF Clone – RC227451L4 OTI Disclaimer: The molecular sequence of this clone aligns with the gene accession number as a point of reference only. However, individual transcript sequences of the same gene can differ through naturally occurring variations (e.g. polymorphisms), each with its own valid existence. This clone is substantially in agreement with the reference, but a complete review of all prevailing variants is recommended prior to use. More info OTI Annotation: This clone was engineered to express the complete ORF with an expression tag. Expression varies depending on the nature of the gene. RefSeq: NM_001145295.1, NP_001138767.1 RefSeq Size: 2421 bp RefSeq ORF: 1677 bp Locus ID: 55768 UniProt ID: Q96IV0, Q96IV0-3 MW: 63.8 kDa Gene Summary: This gene encodes an enzyme that catalyzes hydrolysis of an N(4)-(acetyl-beta-D- glucosaminyl) asparagine residue to N-acetyl-beta-D-glucosaminylamine and a peptide containing an aspartate residue.
    [Show full text]
  • Nelfinavir Inhibits the TCF11/Nrf1-Mediated Proteasome Recovery Pathway in Multiple Myeloma
    cancers Article Nelfinavir Inhibits the TCF11/Nrf1-Mediated Proteasome Recovery Pathway in Multiple Myeloma Dominika Fassmannová 1,2,3 , František Sedlák 1,2,3,4, JindˇrichSedláˇcek 1,2, Ivan Špiˇcka 3,4 and Klára Grantz Šašková 1,2,* 1 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 16610 Prague, Czech Republic; [email protected] (D.F.); [email protected] (F.S.); [email protected] (J.S.) 2 Department of Genetics and Microbiology, Charles University, Viniˇcná 5, 12843 Prague, Czech Republic 3 First Faculty of Medicine, Charles University, Kateˇrinská 32, 12108 Prague, Czech Republic; [email protected] 4 1st Department Medicine—Department of Hematology, Charles University, U Nemocnice 2, 12808 Prague, Czech Republic * Correspondence: [email protected]; Tel.: +420-220-183-518 Received: 5 April 2020; Accepted: 23 April 2020; Published: 25 April 2020 Abstract: Proteasome inhibitors are the backbone of multiple myeloma therapy. However, disease progression or early relapse occur due to development of resistance to the therapy. One important cause of resistance to proteasome inhibition is the so-called bounce-back response, a recovery pathway driven by the TCF11/Nrf1 transcription factor, which activates proteasome gene re-synthesis upon impairment of the proteasome function. Thus, inhibiting this recovery pathway potentiates the cytotoxic effect of proteasome inhibitors and could benefit treatment outcomes. DDI2 protease, the 3D structure of which resembles the HIV protease, serves as the key player in TCF11/Nrf1 activation. Previous work found that some HIV protease inhibitors block DDI2 in cell-based experiments.
    [Show full text]
  • Supplementary Information Patients and Sample Preparation
    Supplementary Information Transcriptome characterization by RNA sequencing identifies two major molecular subgroups with clinical relevance in chronic lymphocytic leukemia Patients and Sample Preparation RNA-sequencing studies were performed in CLL samples from 98 patients, 41 had IGHV-unmutated and 54 IGHV-mutated genes (<98% identity), and in 3 the IGHV mutational status could not be determined. The complete clinical and biological data at the moment of sampling and before any treatment were available in 91 of these patients (Table S2). One hundred and twenty four additional CLL patients sequentially included in the International Cancer Genome Consortium (ICGC) CLL project constituted a validation series and were studied by microarray expression profile. The complete clinical and biological data at the moment of sampling and before any treatment were available in 110 of these patients (Table S2). All patients gave informed consent for their participation in the study following the ICGC guidelines. The tumor samples used for RNA-sequencing were obtained from fresh or cryopreserved mononuclear cells. To purify the CLL fraction, samples were incubated with a cocktail of magnetically-labelled antibodies directed against T cells, NK cells, monocytes and granulocytes (CD2, CD3, CD11b, CD14, CD15 and CD56), adjusted to the percentage of each contaminating population (AutoMACS, Miltenyi Biotec). The degree of contamination by non-CLL cells in the CLL fraction was assessed by immunophenotype and flow cytometry and was lower than 5%. Normal samples were obtained from buffy coats from healthy adult donors with an average age of 54 years (ranging from 45 to 61, Table S5). After Ficoll-Isopaque density centrifugation CD19+ B cells were isolated by positive magnetic cell separation by using AutoMACS system (Milteny Biotec, Auburn, CA).
    [Show full text]
  • Chemico-Genetic Discovery of Astrocytic Control of Inhibition in Vivo
    Article Chemico-genetic discovery of astrocytic control of inhibition in vivo https://doi.org/10.1038/s41586-020-2926-0 Tetsuya Takano1 ✉, John T. Wallace1, Katherine T. Baldwin1, Alicia M. Purkey1, Akiyoshi Uezu1, Jamie L. Courtland2, Erik J. Soderblom1,3, Tomomi Shimogori4, Patricia F. Maness5,6, Received: 23 January 2020 Cagla Eroglu1,2 ✉ & Scott H. Soderling1,2 ✉ Accepted: 19 August 2020 Published online: xx xx xxxx Perisynaptic astrocytic processes are an integral part of central nervous system Check for updates synapses1,2; however, the molecular mechanisms that govern astrocyte–synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte–neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We fnd that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infltration by astrocytic processes. Furthermore, we show that astrocytic NRCAM interacts transcellularly with neuronal NRCAM coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NRCAM reduces numbers of inhibitory synapses without altering glutamatergic synaptic density. Moreover, loss of astrocytic NRCAM markedly decreases inhibitory synaptic function, with minor efects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons and reveal how astrocytes control GABAergic synapse formation and function. The majority of central nervous system (CNS) synapses are ensheathed (Extended Data Fig. 1a), the biotinylation activity of Split 1-TurboID by tiny astrocytic processes1,2.
    [Show full text]